Abstract
Purpose: :
Choroideremia (CHM) is X-linked disease caused by mutations in the Rab Escort Protein 1 gene (REP1) which mediates post-translational isoprenyl modification of newly synthesized Rab GTPases. Rab proteins are the key regulators of vesicular trafficking including phagosome fusion during process of phagocytosis. The purpose of this study was to analyze the effect of depleting of Rep-1 protein in RPE cells on different stages of photoreceptor outer segment (POS) phagocytosis.
Methods: :
The Rep-1 expression was knocked down by transfecting human fetal RPE (hfRPE) cell monolayers with CHM (Rep-1) siRNA using ON-TARGETplus siRNA pool and DharmaFECT 4 Transfection Reagent (Dharmacon, Lafayette, CO). The efficiency of silencing was determined by Western Blot (WB) analysis of Rep-1 protein expression. The phagocytosis assay was performed by challenging hfRPE cells with pHrodo and FITC (Invitrogene,Carlsbad,CA) labeled POS and evaluation of phagocytosis activity by confocal microscopy and using an automated fluorescence microplate reader. Intracellular localization and redistribution of some Rab proteins in response to POS internalization were determined by immunofluorescence and WB analysis of hfRPE transfected with Rep-1 siRNA versus control cells using primary antibodies to Rab 5a, Rab 8a, Rab 11a, Rab 27a and other membrane markers.
Results: :
Transient transfection with siRNA complementary to the coding region of human Rep-1 reduced protein level of Rep-1 by 75%-80% when compared with transfection with non-targeting siRNA. Fluorescence microscopy of Rep-1 depleted hfRPE cells after phagocytic challenge with pHrodo-POS revealed fewer internalized particles compared to non-target siRNA treated hfRPE cells after 5h of POS challenge. Microfluorometric quantification of POS phagocytosis showed that REP-1 silencing resulted in decreased rates of POS phagosome internalization and processing during 2 hours of pulse and up to 26 hours of chase.
Conclusions: :
Our preliminary findings suggest that loss of function mutations in REP-1 could lead to impairment in the process of POS phagocytosis on the stage of phagosome internalization and processing.
Keywords: retinal degenerations: cell biology • retinal pigment epithelium • phagocytosis and killing